Antimineralocorticoid 11b-Substituted Spirolactones Exhibit Androgen Receptor Agonistic Activity: A Structure Function Study

In humans, spironolactone and mespirenone are well known antimineralocorticoids without C-11b substituents. These compounds display antagonist properties by acting through the human androgen receptor (hAR). In contrast, we demonstrate here that synthetic mineralocorticoid antagonists bearing hydrophobic C-11b substituents and C-17g-lactone are potent hAR agonists in vitro. The three-dimensional construction of both the ligand binding domain (LBD) of the hAR and the human mineralocorticoid receptor (hMR), based on the crystal structure of the LBD of the human progesterone receptor, revealed not only that the interactions with the steroidal Aand D-rings seemed to be crucial for stabilization of active hMR or hAR conformation, but that other steroidal substitutions could influence the agonist versus antagonist activity of ligands. The docking of synthetic compounds bearing C-11b hydrophobic substituents within the ligand binding pocket of hAR demonstrated that precise positions of the steroid, such as C-11 and C-17, are in close contact with some residues on the receptor, C-11 with Gly 708 and C-17 with Asn705 and Thr877. These contacts are crucial for the stabilization of the active receptor conformation. Mutation of Asn705 by alanine altered the 11bsubstituted spirolactone-mediated trans-activation function of hAR, suggesting an anchoring of the C-17-lactone carbonyl group (C-22) with this residue. The stabilizing effect of the H12 helix in its active conformation is also induced by hydrophobic contacts between the Gly708 and C-11b substituents, as recently observed with the A773G-hMR mutant in the presence of similar drugs. The study of the role of these substituents suggests efficient new directions for the drug design of selective androgen agonists. Mineralocorticoid and androgen receptors are members of the nuclear receptor superfamily of ligand-responsive transcription factors. They have marked structural and functional similarities (Evans, 1988) and are characterized by three basic functional domains: the amino-terminal A/B domain, which has a ligand-independent trans-activation function; the DNA binding domain, which allows DNA binding and receptor dimerization; and the ligand binding domain (LBD), involved in chaperone protein interaction, dimerization, and hormone-dependent trans-activation. The carboxyterminal regions of these two receptors are relatively well conserved (50% homology). Aldosterone, a natural hormone, exerts its effect through the MR. The action of aldosterone is inhibited by spironolactone and progesterone, which act as competitive inhibitors by forming inactive ligand-receptor complexes. The antimineralocorticoid spirolactones have been used for the past 30 years in the treatment of sodiumretaining states and as antihypertensive agents (Corvol et al., 1981). However, the well known aldosterone antagonist, spironolactone, also displayed such side effects as antiandrogenic activity in rats and humans (Corvol et al., 1975). New antimineralocorticoids have been developed (Singh et al., 2000) in an attempt to obtain derivatives with antagonistic activity higher than that of spironolactone and devoid of the side effects exhibited by this compound (i.e., gynecomastia, decreased libido, and impotence in male patients) due to its antiandrogenic properties (Losert et al., 1986). The actions of the two predominant natural androgens, testosterone and dihydrotestosterone, are mediated through the AR. Compounds like cyproterone acetate or hydroxyflutamide that block the action of androgens have been proved useful in the This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association pour la Recherche sur le Cancer (ARC no. 5205: 1999), and the Fondation pour la Recherche Médicale (FRM). This work was presented as a poster at the 14th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology; Quebec, Canada; 2000 June. ABBREVIATIONS: LBD, ligand binding domain; MR, mineralocorticoid receptor; AR, androgen receptor; hMR, human mineralocorticoid receptor; hAR, human androgen receptor; hPR, human progestin receptor; LBP, ligand binding pocket; PALM, PC-3 androgen luciferase; MMTV, mouse mammary tumor virus; CDTA, trans-1,2-diaminocyclohexane-N,N,N9,N9-tetraacetic acid; DMEM, Dulbecco’s modified Eagle’s medium; WT, wild-type. 0026-895X/01/5905-1307–1313$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 656/900572 Mol Pharmacol 59:1307–1313, 2001 Printed in U.S.A. 1307 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from treatment of benign prostate hypertrophy and prostate cancer in men, or hirsutism in women. To investigate how agonists and antagonists interact with the human MR (hMR) and/or the human AR (hAR), threedimensional models of the hMR and hAR-LBD, have been recently drawn (Fagart et al., 1998; Poujol et al., 2000). They were based on the crystal structure of the holo human retinoic acid receptor-g-LBD (Renaud et al., 1995), human estrogen receptor-a (Brzozowski et al., 1997) and hPR (Williams and Sigler, 1998). The crystal structure of hAR-LBD in complex with the agonist ligand R1881 (methyltrienolone) has recently been determined (Matias et al., 2000) and confirms our model (Poujol et al., 2000). These hMR and hAR-LBD models revealed LBPs, which were further analyzed to identify the residues, involved in the specific recognition of mineralocorticoid or androgen ligands or their antimineralocorticoid or antiandrogen counterparts. We recently showed on hMR that C-11-substituted steroids with a C-17g-lactonic ring displayed antagonist properties, but acted as potent agonists when Ala773 was substituted by Gly (Auzou et al., 2000). Moreover, hAR has a glycine at the corresponding position (Gly708). This residue, when mutated by an alanine, is associated to a partial androgen insensitivity syndrome (Albers et al., 1997). In the present study, the same C-11-substituted steroids with a C-17g-lactonic ring (Fig. 1) were assayed for their capacity to bind and stimulate transcription of hAR. As for Ala773Gly in hMR (Auzou et al., 2000), we determined the specific role of the C-11 substituents facing the 708 glycine residue in the hAR-LBP. We observed that the introduction of an unsaturated hydrophobic group located on the C-11b position of the C-ring does not modify the affinity of these compounds for the hAR and induces potent hAR transcriptional activity in two cellular models: the naive cell line CV-1 or the prostate cancer cell line PC-3 (Térouanne et al., Fig. 1. Chemical structures of steroidal ligands. 1308 Nirdé et al. at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from 2000). We showed that Asn705 is specifically involved in anchoring of the C-22 carbonyl group and Thr877 in anchoring of the 17b-oxygen of the g-lactonic ring. This study clearly demonstrated that this novel family of mineralocorticoid antagonists bearing hydrophobic C-11 substituents and C-17g-lactone acted as potent androgen agonists. Materials and Methods Chemicals. Spironolactone (SC9420) was obtained from Searle Laboratories (Chicago, IL). Mespirenone was from Schering Laboratories (Kenilworth, NJ). [H]R1881 (87 Ci/mmol) and unlabeled R1881 were purchased from NEN Life Science Products (Paris, France). 11b-Vinyl3-oxo-19-nor-17a-pregna-4,9-diene-21,17-carbolactone (1) (Nickisch et al., 1985), 11b-allenyl-3-oxo-19-nor-17a-pregna-4,9-diene-21,17-carbolactone (2), 11-ethylidene-3-oxo-19-nor-17a-pregna-4,9-diene-21,17carbolactone (3), 11-(3-propenylidene)-3-oxo-19-nor-17a-pregna-4,9-diene21,17-carbolactone (4), 11b-(3-hydroxypropyl)-3-oxo-19-nor-17apregna-4,9-diene-21,17-carbolactone (5), 11b-allenyl-3-oxo-19-nor-17apregna-4,9-diene-3,17-dione (6), androsta-4,9-diene-3,17-dione (7), and potassium 3-(17b-hydroxy-3-oxo-19-nor-4,9-diene-17a-yl)-propionate (1-propionate) were synthesized in our laboratory (Faraj et al., 1990; Claire et al., 1993). Structures and abbreviations of the steroids are